Rheological Comparison of Red Cells from Individuals with SCD Treated with Gene Therapy, Haploidentical Transplantation or Hydroxyurea

Background:

Sickle cell disease is caused by point mutation in the beta-globin gene, (HBB) resulting in rigid, dense red blood cells (RBC) that sickle under hypoxic conditions. Outcomes from matched sibling donors (MSD) are excellent, but donor availability is limiting. Patients without a MSD may consider hydroxyurea (HU), haploidentical hematopoietic cell transplant (HaploHCT), or gene therapy (GT). Two GT strategies are FDA approved; a lentiviral gene addition approach that introduces a normal copy of the HBB gene, and a CRISPR/Cas9 strategy that induces fetal hemoglobin (HbF) through disruption of the erythroid enhancer element of the globin gene transcriptional regulator BCL11A(CTX001). Our goal is to assess the red blood cell (RBC) quality of different gene therapy strategies (FDA approved and in clinical trials) and compare to rheology of HaploHCT and to optimal medical management in a cohort of individuals with an excellent HbF response to HU.

Methods:

Individuals treated on NCT05329649 (CTX001), NCT04293185 (BB305), and HU treated individuals provided samples under an Emory IRB approved protocol in EDTA tubes. NCT04443907 (OTQ923) and NCT04819841 clinical trial samples were provided by the sponsor. HU samples were obtained from individuals at maximum tolerated dose exhibiting a final %HbF of 20% or more, without hereditary persistence of fetal hemoglobin. HaploHCT samples were collected under a St Jude Children’s Research Hospital sponsored clinical trial (NCT04362293). All samples were collected without exogenous HbA present, 6-12 months after graft infusion; 18 months for NCT04819841. Complete blood counts (CBC) were obtained on an ADVIA hematology analyzer which provides percent dense red blood cells (%DRBC). %HbF was measured using high performance liquid chromatography (HPLC). Oxygen gradient ektacytometry (LoRRca) was used to assess red cell deformability; readouts are elongation index minimum (EIMin) deformability under hypoxia, elongation index maximum (EIMax) deformability under normoxia, and point of sickling (PoS), the oxygen tension at which HbS polymerization begins. Kruskal-Wallis or Mann Whitney tests were used for comparisons, and interquartile range (IQR) calculated to assess spread of values within each group.

Results:

We compared the GT (CTX001, n=2, BB305, n=2, and sponsored clinical trial OTQ923, n=4), HaploHCT (n=4), and HU (n=15) groups. EImin, EImax, DRBC, and RBC were significantly different in HU compared to HaploHCT (p: 0.001, 0.021, 0.01, and 0.03, respectively). The median and IQR were as follows: EImax (HU: 0.50(0.47-0.56),GT: 0.57(0.49-0.59), HaploHCT: 0.59(0.59-0.60)), EImin (HU: 0.15 (0.13-0.19),GT:0.29 (0.23-0.37), HaploHCT: 0.59(0.57-0.59)), DRBC (HU:4.9 (3.4-6.3), GT:2.1(1.6-6.6), HaploHCT:0.1(0.1-1.1)), and RBC (HU: 2.8(2.5-3.4), GT:3.4 (3.0-4.0),HaploHCT: 3.9 (3.4-4.1)). The range of red cell function values was narrow for HaploHCT, wide for GT and HU. Significantly lower EImin and EImax in HU compared to HaploHCT indicates worse RBC deformability under normoxia and hypoxia. %HbF levels were comparable between HU and GT. There was no statistically significant difference in PoS between GT and HU; HaploHCT treated individuals’ RBC did not sickle and had no PoS.

Conclusions:

RBC function testing provides an opportunity to compare optimal HU response and curative strategies for SCD, including GT and HaploHCT. We observed a wide range of red cell function values in the GT group, suggesting that with appropriate clinical correlations, these tests can help compare different GT strategies by assessing the degree of red cell correction achieved by each one.

We found that the RBC quality and function of HU was comparable to GT, when samples are selected from individuals having a HbF/functional hemoglobin response comparable to that that of individuals post GT. Post HaploHCT, individuals exhibited nearly normalized RBC rheology, with very similar inter-individual results. Further studies are needed to confirm our findings in a larger SCD patient cohort, including samples pre- and post-transplant; longer follow-up of both strategies are also needed.